WO2012020845A1 - Procédé de production de cellules produisant une hormone pancréatique - Google Patents

Procédé de production de cellules produisant une hormone pancréatique Download PDF

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WO2012020845A1
WO2012020845A1 PCT/JP2011/068487 JP2011068487W WO2012020845A1 WO 2012020845 A1 WO2012020845 A1 WO 2012020845A1 JP 2011068487 W JP2011068487 W JP 2011068487W WO 2012020845 A1 WO2012020845 A1 WO 2012020845A1
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cells
medium
inhibitor
kinase
cell
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PCT/JP2011/068487
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Japanese (ja)
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細谷　昌樹
昌伸 庄司
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武田薬品工業株式会社
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Priority to AU2011290123A priority Critical patent/AU2011290123B2/en
Priority to US13/814,878 priority patent/US9157069B2/en
Priority to NZ607608A priority patent/NZ607608A/en
Priority to SG2013007455A priority patent/SG187655A1/en
Priority to CA2807935A priority patent/CA2807935C/fr
Priority to CN201180048849.0A priority patent/CN103154240B/zh
Application filed by 武田薬品工業株式会社 filed Critical 武田薬品工業株式会社
Priority to KR1020137006027A priority patent/KR101877077B1/ko
Priority to RU2013109949/10A priority patent/RU2576000C2/ru
Priority to JP2012528721A priority patent/JP5875517B2/ja
Priority to EP11816507.5A priority patent/EP2604685B1/fr
Publication of WO2012020845A1 publication Critical patent/WO2012020845A1/fr
Priority to IL224451A priority patent/IL224451A/en

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    • C12N5/0676—Pancreatic cells
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • C12N2501/38—Hormones with nuclear receptors
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Definitions

  • the present invention relates to a method for producing pancreatic hormone-producing cells, a medicament containing pancreatic hormone-producing cells obtained by the production method, and the like.
  • pancreas is an organ that has an endocrine gland (endocrine cell) and an exocrine gland (exocrine cell) and plays an important role in both.
  • Exocrine cells mainly play a role in secreting digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase.
  • Endocrine cells play a role in secreting pancreatic hormones, pancreatic ⁇ cells to glucagon, pancreatic ⁇ cells to insulin, pancreatic ⁇ cells to somatostatin, and PP cells to pancreatic polypeptide (sometimes abbreviated as PP herein). Is known to be secreted. In recent years, it has been reported that ghrelin, a gastric secretion hormone, is also secreted from the pancreas.
  • Insulin plays an important role in promoting glucose utilization, protein synthesis, neutral fat formation and storage, lowering blood sugar levels, and keeping blood sugar at the correct concentration.
  • Pancreatic glucagon plays an important role in the mechanism of regulating glucose metabolism along with insulin as a blood glucose-rising hormone through liver glycogenolysis and gluconeogenesis.
  • Somatostatin exerts its action through binding to the somatostatin receptor and suppresses the secretion of various hormones such as glucagon and insulin in the pancreas.
  • PP is a hormone secreted from cells of the islets of Langerhans in response to meals, and is known as a satiety factor and has a function of reducing food intake and weight gain.
  • Ghrelin is known to increase body weight by stimulating food intake and reducing fat oxidation.
  • Diabetes mellitus is a disease that develops when insulin is deficient or loses its function, and is difficult to cure once it develops. Diabetes can be broadly classified into two types: type I diabetes (insulin-dependent diabetes) and type II diabetes (non-insulin-dependent diabetes).
  • Type II diabetes is a chronic disease that develops because of resistance to insulin, and is a diabetes that is a problem in relation to lifestyle habits such as obesity and stress caused by overeating and lack of exercise. Type II diabetes often develops in middle-aged and elderly, and many diabetics are of this type.
  • Type I diabetes is a chronic disease that occurs when insulin-producing cells are destroyed by autoimmune disease or viral infection, and insulin is not secreted into the body.
  • Pancreatic transplantation or islet transplantation is performed for type I diabetic patients as a treatment that can automatically control blood glucose levels that constantly change in the body and reduce the burden on the patient. Although it is possible to achieve normal blood glucose levels with these treatments, transplantation techniques are not well established, and there are currently insufficient transplantable pancreas or islets . In addition, in order to avoid immune rejection to the graft, the patient needs to continue to take the immunosuppressant for a lifetime, and problems such as the risk of infection and side effects due to the immunosuppressant remain.
  • One of the treatments that have been tried for type I diabetes is to regenerate and transplant the patient's insulin-producing cells themselves. This method can produce insulin in the patient's own body. In addition, since it is a patient-derived cell, the problem of rejection is solved, which is advantageous in terms of safety.
  • Known methods for obtaining insulin-producing cells include a method of differentiating from ES cells, a method of differentiating from tissue stem cells of a patient's pancreas, a method of extracting and differentiating a patient's pancreatic duct epithelium-derived cells outside the body, and the like.
  • a method of inducing differentiation of pancreatic ⁇ cells from human ES cells using activin or retinoic acid (RA) (Patent Document 1, Non-Patent Documents 1 to 4) or glucagon producing cells ( ⁇ cells) from human ES cells )
  • RA retinoic acid
  • Non-Patent Document 8 pancreatic ⁇ cell differentiation induction from human iPS cells
  • ES cells are important transcription factors involved in pancreatic development
  • insulin-producing cells obtained by these methods have considerably lower insulin production efficiency than normal pancreatic ⁇ cells, and development of a method for efficiently obtaining insulin-producing cells applicable to cell medical applications is still in progress. It has been demanded. Furthermore, there is a demand for increasing the number of cells obtained to a practical level for treating diabetes.
  • An object of the present invention is to provide a method for producing pancreatic hormone-producing cells more suitable for application of cell therapy, a medicament containing pancreatic hormone-producing cells obtained by the production method, and a method for screening a therapeutic agent for diabetes using the cells. It is to be.
  • pancreatic hormone-producing cells that mimic the development of pancreas (a form that maintains a three-dimensional structure) can be produced from stem cells, and the present invention has been completed.
  • a method for producing pancreatic hormone-producing cells characterized in that the stem cells are subjected to the following steps (1) to (6): (1) Step of culturing stem cells in medium containing Rho kinase inhibitor (2) Step of culturing cells obtained in (1) above in medium containing GSK3 inhibitor (3) Obtained in (2) above A step of culturing the obtained cells in a medium containing a GSK3 inhibitor and an activin receptor-like kinase-4,7 activator (4) After forming a cell mass from the cells obtained in (3) above (5) culturing the cell mass in a suspended state in a medium (5) The cells obtained in the above (4) are transformed into retinoic acid receptor agonist, AMP-activated protein kinase and / or activin receptor-like kinase-2, A step of culturing in a medium containing an inhibitor of 3,6, an inhibitor of activin receptor-like kinase-4,5,7, and a cell growth
  • Rho kinase inhibitor in step (1) is (+)-(R) -trans-4- (1-aminoethyl) -N- (4-pyridyl) cyclohexanecarboxamide dihydrochloride.
  • the production method according to the above [1] or [2]; [4] The GSK3 inhibitor in steps (2) and (3) is (I) 6-[[2-[[4- (2,4-Dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] ethyl] amino] nicotinonitrile And / or (ii) (2′Z, 3′E) -6-bromoindirubin-3′-oxime, The production method according to any one of the above [1] to [3]; [5] The retinoic acid receptor agonist in step (5) is retinoic acid, The production method according to any one of [1] to [4] above; [6] The inhibitor of AMP-activated protein kinase and / or activin receptor-like kinase-2, 3, 6 in step (5) is dosomorphin.
  • the inhibitor of activin receptor-like kinase-4,5,7 in step (5) is 4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide,
  • the cell growth factor in the step (5) is a basic fibroblast growth factor.
  • iPS cell induced pluripotent stem cell
  • ES cell embryonic stem cell
  • pancreatic hormone-producing cells are
  • a method for producing pancreatic hormone-producing cells wherein the endoderm cells are subjected to the following steps (4 ′) and (5 ′); (4 ′) a step of forming a cell mass from endoderm cells and culturing the cell mass in a suspended state in a medium (5 ′) the cell obtained in (4 ′) above is treated with a retinoic acid receptor agonist, AMP Culturing in a medium containing an activated protein kinase and / or an inhibitor of activin receptor-like kinase-2,3,6, an inhibitor of activin receptor-like kinase-4,5,7, and a cell growth factor; [14] A medicament comprising pancreatic hormone-producing cells obtained by the production method according to any one of [1] to [13] above; [15] A screening method for a therapeutic agent for diabetes, comprising using cells obtained by any one or more steps selected from the group consisting of the following steps (1) to (6): (1) Step of culturing stem cells in
  • pancreatic hormone-producing cells that mimic pancreas development can be produced from stem cells.
  • the cells obtained by any one or more of the steps (1) to (6) are useful for the prevention and / or treatment of diseases caused by pancreatic hormone production and / or secretion abnormalities such as diabetes. It can be used for screening of compounds.
  • the cells of the present invention can be used for cell therapy for treating such diseases and maintain a three-dimensional structure, they are compared with pancreatic hormone-producing cells obtained by conventional production methods. However, it is a more suitable cell for application to cell medicine.
  • Induction of differentiation from human iPS cells was initiated using various factors, and the expression of primitive streak marker (Brachyury) and endoderm marker (SOX17) during the first 4 days was measured daily by quantitative RT-PCR. Results are shown.
  • the expression level of each gene was shown as a relative value to the expression level of GAPDH, which is a housekeeping gene.
  • the expression level of Brachyury was transiently increased on the third day of culture, and the expression level of SOX17 was significantly increased on the fourth day.
  • the nuclei of SOX17 positive cells are green with Alexa488 (indicated as SOX17 in the figure), and the nuclei of cells are blue with Hoechst 33342 (indicated as Hoechst in the figure).
  • staining image is shown as Merge. It was observed that most cells expressed SOX17 protein.
  • Cells that have been induced to differentiate for 4 days from human iPS cells using activin A and CHIR99021 are seeded on a 96-well spheroid plate and cultured for 1 day, and then contain 1% B27 supplemented with dosomorphin, retinoic acid, SB431542 and bFGF
  • the result of the expression analysis of insulin when it is cultured in Improved MEM Zinc Option medium for 8 days and then replaced with Improved MEM Zinc Option medium containing 1% B27 is shown (shown as Insulin in the figure).
  • the gene expression level was expressed as a relative value to the expression level of GAPDH, which is a housekeeping gene.
  • Insulin expression increased over time until the 23rd day of culture after replacing the Implanted MEM Zinc Option medium containing 1% B27 on the 13th day of culture.
  • Cells that have been induced to differentiate for 4 days using activin A and CHIR99021 from human iPS cells are seeded on a 96-well spheroid plate and cultured for 1 day, and then a combination of dosomorphin, retinoic acid, SB431542 and bFGF, or dosomorphin, retinoin Culture is performed for 8 days using a combination of acid and SB431542, followed by culturing in Improved MEM Zinc Option medium containing 1% B27, and the result of expression analysis of insulin on the 19th day is shown (shown as Insulin in the figure).
  • the gene expression level was expressed as a relative value to the expression level of GAPDH, which is a housekeeping gene.
  • the amount of insulin expressed on the 19th day of culture was higher when cultured with a combination of dosomorphin, retinoic acid, SB431542 and bFGF than when cultured with a combination of dosomorphin, retinoic acid and SB431542.
  • FIG. 5 shows the result of preparing a frozen section using the cell sphere on the 21st day of culture induced differentiation in the same manner as in the method of FIG. 4 and immunofluorescent staining using an anti-insulin antibody and an anti-glucagon antibody.
  • Insulin positive cells are red with Alexa568 (indicated as Insulin in the figure), glucagon positive cells are green with Alexa488 (in the figure, indicated as Glucagon), and the cell nucleus is blue with Hoechst 33342 (in the figure, Hoechst). ). Moreover, the figure which synthesize
  • BD matrigel or fibronectin is used as a substrate to induce endoderm from human iPS cells using activin A and CHIR99021, to form a cell sphere, and then to pancreatic hormone-producing progenitor cells, followed by pancreatic hormone-producing cells
  • the result of the expression analysis of various differentiation markers when differentiation-inducing to is shown.
  • the expression level of each gene was shown as a relative value to the expression level of GAPDH, which is a housekeeping gene.
  • SOX17 expression decreased markedly with differentiation, and PDX1 and NGN3 expression gradually increased until day 17 of culture. Insulin expression increased rapidly from day 17 of culture.
  • pancreatic hormone-producing cell means a cell having the ability to produce pancreatic hormones.
  • the pancreatic hormone-producing cells do not always have to produce pancreatic hormones, as long as they have pancreatic hormone-producing ability. Therefore, the amount of pancreatic hormone produced is not particularly limited. Examples of pancreatic hormones include insulin, glucagon, somatostatin, pancreatic polypeptide, and ghrelin.
  • Pancreatic hormone producing cells include insulin producing cells (synonymous with pancreatic ⁇ cells), glucagon producing cells (synonymous with pancreatic ⁇ cells), somatostatin producing cells (synonymous with pancreatic ⁇ cells), pancreatic polypeptide (PP) producing cells, ghrelin producing Cell. Among these, insulin producing cells are preferable.
  • stem cell refers to a cell that can be cultured in vitro and can differentiate into a plurality of cells constituting a living body.
  • embryonic stem cells ES cells
  • pluripotent stem cells derived from embryonic primordial germ cells (EG cells: Proc Natl Acad Sci USA 1998, 95: 13726-31)
  • testis-derived pluripotency Stem cells GS cells: Nature. 2008, 456: 344-9
  • somatic cell-derived induced pluripotent stem cells induced pluripotent stem cells
  • iPS cells somatic stem cells
  • tissue stem cells preferably iPS cells, ES cells or human somatic stem cells, more preferably iPS cells.
  • ES cells cells derived from any warm-blooded animal, preferably a mammal, can be used. Examples of mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans. Preferably, cells derived from humans can be used.
  • ES cells such as mammals established by culturing early embryos before implantation, and established by culturing early embryos produced by nuclear transfer of somatic cell nuclei were established. ES cells and ES cells obtained by modifying the genes on the chromosomes of these ES cells using genetic engineering techniques are included.
  • Each ES cell can be prepared according to a method commonly practiced in the art or according to known literature.
  • Mouse ES cells were obtained in 1981 from Evans et al. (1981, Nature 292: 154-6) and Martin et al. (Martin GR. Et al., 1981, Proc Natl Acad Sci 78: 7634-8). For example, it can be purchased from Dainippon Sumitomo Pharma Co., Ltd. (Osaka, Japan).
  • Human ES cells were established in 1998 by Thomson et al. (Science, 1998, 282: 1145-7) and are available at the WiCell Research Institute (WiCell Research Institute, website: http: // www. available from Wisell.org/, Madison, Wisconsin, USA, National Institute of Health, Kyoto University, etc. For example, Cellartis (website: http://www.cellaritis.com) /, Sweden).
  • iPS cells cells derived from any warm-blooded animal, preferably a mammal, can be used.
  • mammals include mice, rats, guinea pigs, hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses, goats, monkeys, and humans.
  • cells derived from humans can be used.
  • iPS cells include cells obtained by introducing a plurality of genes into somatic cells such as skin cells, which have acquired multipotency similar to ES cells (for example, Oct3 / 4 gene, Klf4 gene, c-Myc IPS cells obtained by introducing a gene and Sox2 gene, iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene and Sox2 gene (Nat Biotechnol 2008; 26: 101-106)) and the like.
  • a method in which a transgene is further reduced (Nature. 2008 Jul 31; 454 (7204): 646-50)
  • a method using a low molecular weight compound Cell Stem Cell. 2009 Jan 9; 4 (1): 16 -9, Cell Stem Cell.
  • iPS cell production method has been intensively improved technically, but the basic properties of the produced iPS cell, that is, having pluripotency are the same regardless of the production method, Any of them can be used in the production method of the present invention.
  • the somatic stem cells those derived from humans can be used.
  • the somatic stem cell is a cell that can differentiate into a pancreatic hormone-producing cell, and examples thereof include bone marrow and adipose-derived mesenchymal stem cells and stem cells present in the pancreas.
  • pancreatic hormone-producing cells can be produced from various stem cell lines such as human iPS cell lines having different production methods.
  • the production method of the present invention is a method for producing pancreatic hormone producing cells from stem cells.
  • the production method of the present invention is also a method for inducing differentiation of a cell (stem cell) in a more undifferentiated state into a more differentiated state (pancreatic hormone-producing cell).
  • the production method of the present invention includes the following steps (1) to (6).
  • the cells obtained in the above (4) are retinoic acid receptor agonist, AMP-activated protein kinase and / or activin receptor-like kinase-2, A step of culturing in a medium comprising an inhibitor of 3, 6; an inhibitor of activin receptor-like kinase-4, 5, 7; and a cell growth factor (6) culturing the cell obtained in (5) above in the medium Process
  • Step (1) A step of culturing a stem cell in a medium containing a Rho kinase inhibitor This step is a pre-stage of the step of inducing differentiation from a stem cell to a pancreatic hormone-producing cell in Step (2) described later, This corresponds to the stage of preculture (seeding) of stem cells.
  • the stem cells in this step may be those co-cultured with feeder cells or feeder cell extracts.
  • feeder cells refer to cells that provide an environment in which other types of cells can proliferate by co-culture.
  • Stem cells in this step may be either dispersed cells or non-dispersed cells, but are desirably dispersed cells.
  • As the dispersed cells a single cell and a cell mass composed of several (typically about 2 to 500, 20 to 200, or 50 to 100) cells [as described in the step (4) described below, Cell mass refers to a cell in which a plurality of cells are bonded to each other to form a single mass.] In this step, the cell mass is a cell mass. desirable.
  • Preparation of dispersed cells can be performed by a method known per se. Examples of such a method include operations such as treatment with a chelating agent (eg, EDTA), an enzyme (eg, trypsin, collagenase), and mechanical dispersion (eg, pipetting).
  • a chelating agent eg, EDTA
  • an enzyme eg, trypsin, collagenase
  • mechanical dispersion eg, pipetting
  • Dispersed cells are floating cells (floating cells are cells that are not attached to the incubator or substrate), or adherent cells (adherent cells are those that are attached to the incubator or substrate. Cell).
  • this step after removing the feeder cells or the feeder cell extract from the above-mentioned stem cells (for example, by removing the supernatant by removing it from the centrifuge tube and leaving it for 2 to 10 minutes, and removing the supernatant) It is desirable to culture in a medium containing an inhibitor (that is, to induce differentiation without using feeder cells from the time of seeding).
  • Rho kinase inhibitor refers to a substance that inhibits the activity of Rho kinase.
  • Rho kinase is a kind of low molecular weight GTP binding protein (low molecular weight G protein) included in the category of GTPase which is a degrading enzyme of GTP (guanosine triphosphate). It has a coiled-coil region at the part and a Rho interaction region at the carboxy terminus (Amano et al., Exp. Cell. Res., 261, 44-51 (2000)).
  • Rho kinase inhibitor examples include 1- (5-isoquinolinesulfonyl) -2-methylpiperazine (H-7), 1- (5-isoquinolinesulfonyl) -3-methylpiperazine (isoH-7).
  • Rho kinase inhibitors can be used.
  • the stem cells are cultured in a medium containing a Rho kinase inhibitor.
  • concentration of the Rho kinase inhibitor in the medium is not particularly limited as long as it can achieve a desired effect such as improvement of the survival rate of stem cells, but is usually 0.01 to 1000 ⁇ M, preferably 0.1. It is ⁇ 100 ⁇ M, particularly preferably 1.0-50 ⁇ M.
  • Y-27632 is used as a Rho kinase inhibitor, it can be used at a concentration of preferably about 1.0 to about 30 ⁇ M, more preferably about 2.0 to about 20 ⁇ M.
  • Fasudil / HA1077 When Fasudil / HA1077 is used as a Rho kinase inhibitor, it can be used at a concentration about twice that of Y-27632. When a plurality of types of Rho kinase inhibitors are used in combination, each inhibitor is appropriately increased or decreased based on the above concentration range.
  • the culture time in the medium containing the Rho kinase inhibitor is not particularly limited as long as it is a length of time that can achieve a desired effect such as improvement of the survival rate of stem cells.
  • the desired effect can be obtained by culturing in a medium containing a Rho kinase inhibitor for about 12 hours or more (eg, 12 to 72 hours) after the human iPS cells are dispersed. You can get enough.
  • the density of the stem cells in the medium containing the Rho kinase inhibitor is not particularly limited as long as it can achieve a desired effect such as improvement of the survival rate of the stem cells.
  • the medium used in this step is not particularly limited as long as it contains a Rho kinase inhibitor.
  • a Rho kinase inhibitor is added to a medium used for culturing stem cells (hereinafter also referred to as a basal medium for convenience). It is added.
  • the basal medium used in this step includes primate ES / iPS cell medium (reprocell medium), BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle A MEM medium, an ⁇ MEM medium, a DMEM medium, a ham medium, an RPMI 1640 medium, a Fischer's medium, a medium obtained by mixing two or more kinds of media arbitrarily selected from these media, and the like can be used.
  • the medium is not particularly limited as long as it can be used for culturing animal cells.
  • reprocell medium these media can be purchased from Reprocell, Invitrogen, SIGMA, Cosmo Bio.
  • a medium substantially free of serum and / or serum extract is preferable, and a serum-free medium is more preferable.
  • the term “substantially free of serum” means that the serum content is less than about 1% by volume, preferably less than about 0.1% by volume, more preferably less than about 0.01% by volume. means.
  • a serum-free medium means a medium that does not contain unadjusted or unpurified serum, and a medium that contains purified blood-derived components or animal tissue-derived components (for example, growth factors) corresponds to a serum-free medium. To do.
  • the medium used in this step may contain a serum substitute.
  • serum substitutes include albumin (eg, lipid-rich albumin), transferrin, fatty acid, collagen precursor, trace elements (eg, zinc, selenium), B27 supplement, N2 supplement, knockout sealant replacement, 2-mercaptoethanol, 3 'thiol glycerol, and equivalents thereof.
  • Knockout sealum replacement can be purchased from Invitrogen.
  • Other serum substitutes can be purchased from Invitrogen, SIGMA, Wako Pure Chemical Industries, Sumitomo Dainippon Pharma, and the like.
  • the concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight.
  • the medium used in this step is preferably a medium that substantially does not contain feeder cells and / or feeder cell extracts, and more preferably a medium that does not contain any feeder cells and / or feeder cell extracts. preferable.
  • the term “substantially free of feeder cells and / or feeder cell extract” means that the content of the feeder cells and / or feeder cell extract in the medium is less than about 5% by volume, preferably about 1% by volume. Less than, more preferably less than about 0.01% by volume.
  • the pancreatic hormone-producing cells produced by the production method of the present invention are contaminated with substances that cause rejection (eg, animal-derived cells). Less is.
  • the culture in this step is usually performed using an incubator.
  • an incubator is not particularly limited as long as it can cultivate stem cells, but is desirably cell-adhesive when performing adhesion culture, and is non-cell-adhesive when performing suspension culture. Is desirable.
  • Examples of incubators include flasks, tissue culture flasks, dishes, petri dishes, tissue culture dishes, multi dishes, micro plates, micro well plates, multi plates, multi well plates, micro slides, chamber slides, petri dishes, tubes , Trays, culture bags, and roller bottles.
  • the cell-adhesive incubator is coated with an arbitrary cell-supporting substrate such as an extracellular matrix (ECM) for the purpose of improving adhesion with cells on the surface of the incubator.
  • ECM extracellular matrix
  • Examples of the incubator used for adhesion culture include dishes, flasks, microplates, and cell culture sheets. These incubators are coated with a substrate for supporting cells such as collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, etc. to improve adhesion to cells. May be.
  • the cell culture sheet refers to a support for culturing cells in a sheet form, and for example, OptiCell (Nunc) is commercially available.
  • OptiCell As the cell support substrate in this step, Type I-collagen, BD Matrigel (Nippon Becton Decktonson), fibronectin (Invitrogen), etc.
  • the incubator used for suspension culture include dishes, flasks, microplates, tubes, and roller bottles. These incubators are made of a hydrophobic material or coated with a material that prevents adsorption of cells and proteins, such as hydrogel and lipid. In order to efficiently form cell clumps, it is desirable to use an incubator having a U-shaped or V-shaped bottom surface.
  • the culture temperature is not particularly limited as long as it is suitable for culturing the stem cells to be used, and may be about 30 to 40 ° C., preferably about 37 ° C.
  • the CO 2 concentration can be about 1-10%, preferably about 2-5%.
  • the oxygen partial pressure can be 1-10%.
  • Step (2) A step of culturing the cells obtained in the step (1) in a medium containing a GSK3 inhibitor. ) And the step of inducing differentiation from stem cells to endoderm cells.
  • GSK3 glycogen synthase kinase 3
  • GSK3 serine / threonine protein kinase
  • GSK3 has GSK3 ⁇ and GSK3 ⁇ isoforms encoded by different genes and having high homology at the amino acid level. It is also known that GSK3 is also involved in the Wnt signal, and that Wnt signal is activated by inhibiting GSK3.
  • the GSK3 inhibitor include a GSK3 ⁇ inhibitor and a GSK3 ⁇ inhibitor. In this step, a GSK3 ⁇ inhibitor is desirable.
  • GSK3 inhibitors are either commercially available or can be synthesized according to previous reports.
  • Methods using Wnt-3A peptide for inducing differentiation from stem cells to endoderm cells are known (Non-Patent Documents 1, 2, and 5).
  • excellent operability, reproducibility, and selectivity can be provided by using a GSK3 inhibitor that is a low molecular compound.
  • the GSK3 ⁇ inhibitor is preferably CHIR99021 (6-[[2-[[4- (2,4-dichlorophenyl) -5- (4-methyl-1H-imidazol-2-yl) -2-pyrimidinyl] amino] Ethyl] amino] nicotinonitrile) or BIO ((2′Z, 3′E) -6-bromoindirubin-3′-oxime).
  • BIO ((2′Z, 3′E) -6-bromoindirubin-3′-oxime).
  • the concentration of the GSK3 inhibitor in the medium is appropriately set depending on the type of the inhibitor to be used, but is usually 0.01 to 100 ⁇ M, preferably 0.1 to 10 ⁇ M.
  • CHIR99021 it is usually 0.1-20 ⁇ M, preferably 1-5 ⁇ M, and in the case of BIO, it is usually 0.01-5 ⁇ M, preferably 0.1-2 ⁇ M.
  • each inhibitor is appropriately increased or decreased based on the above concentration range.
  • the medium used in this step is not particularly limited as long as it contains a GSK3 inhibitor, and is usually obtained by adding a GSK3 inhibitor to a medium (basic medium) used for culturing stem cells.
  • a medium used for culturing stem cells.
  • the basal medium used in this step for example, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM ZincOption medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, serum-free DMEM / F12 medium, ham medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof.
  • the basal medium used in this step is not particularly limited as long as it can be used for culturing animal cells.
  • the basal medium used in this step is preferably serum-free DMEM / F12 medium, RPMI 1640 medium, Improved MEM Zinc Option medium, and particularly preferably serum-free DMEM / F12 medium.
  • a medium substantially free of serum and / or serum extract is preferable, and a serum-free medium is more preferable.
  • feeder cells and / or feeder cell extracts are not substantially used in this step, the pancreatic hormone-producing cells produced by the production method of the present invention are contaminated with substances that cause rejection (eg, animal-derived cells). Less is.
  • the medium used in this step may contain a serum substitute.
  • Serum replacements include albumin, transferrin, fatty acids, collagen precursors, trace elements (eg zinc, selenium), B27 supplements, N2 supplements, knockout serum replacement, 2-mercaptoethanol, 3'thiol glycerol, and equivalents thereof Such as things.
  • B27 supplement is preferable.
  • the concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight.
  • These serum substitutes can be purchased from Invitrogen, SIGMA, Wako Pure Chemical Industries, Dainippon Sumitomo Pharma and the like.
  • the culture temperature in this step is not particularly limited as long as it is suitable for culturing the stem cells to be used.
  • the culture time is 6 to 144 hours, preferably 12 to 72 hours, at a culture temperature of about 37 ° C.
  • the culture in this step is usually performed in an incubator in which about 1 to 10%, preferably 5% CO 2 is aerated.
  • Step (3) A step of culturing the cells obtained in the step (2) in a medium containing a GSK3 inhibitor and an activin receptor-like kinase-4,7 activator. This step is performed subsequent to the above, and corresponds to a step of completing differentiation induction from stem cells into endoderm cells.
  • the activin receptor-like kinase (ALK) -4,7 activator used in this step is selected from substances having an activating action on ALK-4 and / or ALK-7.
  • Examples of activin receptor-like kinase-4,7 activators used in this step include activin, Nodal, and Myostatin, all of which are commercially available. Of these, activin is preferable as the activator of activin receptor-like kinase-4, 7 used in this step.
  • the activin is a 24 kD peptide cell growth / differentiation factor belonging to the TGF ⁇ (transforming growth factor ⁇ ) family, and two ⁇ subunits constitute a dimer via SS bonds ( Ling, N., et al., (1986) Nature 321, 779-782; Vale, W., et al., (1986) Nature 321, 776-779).
  • any of activins A, B, C, D, and AB, and those derived from any animal such as human and mouse can be used, and these are commercially available. Of these, activin A is particularly preferably used.
  • Activins derived from the same animal species as the animal species from which the stem cells are used are preferably used.
  • human activin A is preferably used.
  • the concentration of the activin receptor-like kinase-4,7 activator in the medium in this step is appropriately set depending on the type of activin receptor-like kinase-4,7 activator used.
  • the concentration in the medium is usually 0.1 to 200 ng / ml, preferably 5 to 150 ng / ml, particularly preferably 10 to 100 ng / ml.
  • any one or a combination of two or more activin receptor-like kinase-4,7 activators can be used.
  • this step is characterized in that a GSK3 inhibitor is included in the medium together with an activator receptor-like kinase-4,7 activator. If stem cells are cultured in the presence of activin and a GSK3 inhibitor, they can be differentiated more suitably into endoderm cells.
  • Examples of the GSK3 inhibitor used in this step include a GSK3 ⁇ inhibitor and a GSK3 ⁇ inhibitor.
  • a GSK3 ⁇ inhibitor is preferable.
  • Specific examples of the GSK3 inhibitor used in this step include those similar to the GSK3 inhibitor exemplified in the above step (2), but also in this step, CHIR99021 or BIO which is a GSK3 ⁇ inhibitor is used.
  • the concentration of the GSK3 inhibitor in the medium is appropriately set depending on the type of the inhibitor used. In the case of CHIR99021, it is usually 0.1 to 20 ⁇ M, preferably 1 to 5 ⁇ M, and in the case of BIO, it is usually 0.01 to 5 ⁇ M.
  • the 0.1 to 2 ⁇ M Preferably, it is 0.1 to 2 ⁇ M.
  • either one kind or a combination of two or more kinds of GSK3 inhibitors can be used.
  • each inhibitor is appropriately increased or decreased based on the above concentration range. And use it.
  • activin receptor-like kinase-4,7 activator and GSK3 inhibitor may be added simultaneously to the medium, and as long as differentiation of stem cells into endoderm cells can be induced, It may be added to the medium with a time difference separately. It is convenient and preferable that the activin receptor-like kinase-4,7 activator and the GSK3 inhibitor are simultaneously added to the medium.
  • the medium used in this step is prepared by adding an activin receptor-like kinase-4,7 activator and a GSK3 inhibitor to the basal medium exemplified in the above step (2).
  • the medium used in this step may be prepared using the same type of basal medium as used in step (2) above, or may be prepared using a different type of basal medium.
  • the basal medium of the same kind eg, serum-free DMEM / F12 medium
  • the basal medium used in this step is preferably serum-free DMEM / F12 medium, RPMI 1640 medium, Improved MEM Zinc Option medium, and particularly preferably serum-free DMEM / F12 medium.
  • substantially no feeder cells and / or feeder cell extracts are used, more preferably no feeder cells and / or feeder cell extracts are used.
  • a medium used in this step a medium substantially free of serum and / or serum extract is preferable, and a serum-free medium is more preferable.
  • the pancreatic hormone-producing cells produced by the production method of the present invention are contaminated with substances that cause rejection (eg, animal-derived cells). Less is.
  • the medium used in this step may contain a serum substitute.
  • Serum replacements include albumin, transferrin, fatty acids, collagen precursors, trace elements (eg zinc, selenium), B27 supplements, N2 supplements, knockout serum replacement, 2-mercaptoethanol, 3'thiol glycerol, and equivalents thereof B27 supplement is preferable.
  • B27 supplement the concentration in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight.
  • the culture temperature in this step is not particularly limited as long as it is a culture temperature suitable for culturing the cells to be used, but is about 30 to 40 ° C, preferably about 37 ° C.
  • the culture time is 6 to 288 hours, preferably 12 to 124 hours at a culture temperature of about 37 ° C.
  • the culture in this step is usually performed in an incubator in which about 1 to 10%, preferably 5% CO 2 is aerated.
  • confirmation that stem cells have been induced to differentiate into endoderm cells is performed using an endoderm marker.
  • an endoderm marker can be carried out by evaluating the presence or absence of expression of a protein or gene (endoderm marker) expressed specifically in the endoderm cells.
  • Protein expression can be evaluated by a method using an antigen-antibody reaction, and gene expression can be evaluated by a method using RT-PCR.
  • the marker include SOX17 (sex determining region Y, Sex determining region Y), Googlesec (googood homebox), CXCR4 (chemokine (C-C-Cmotif) receptor 4), FOXA2 (forkA2, etc.).
  • Step (4) A step of forming a cell sphere from the cells obtained in the step (3) and then culturing the cell mass in a suspended state in a medium.
  • This step comprises the step (3) Step of culturing the cell mass in a suspended state in a medium after forming the cell mass of the endoderm cells obtained in the above, that is, the endoderm cells, (step of re-seeding) It is.
  • the cell mass refers to a state in which a plurality of cells adhere to each other to form one mass (for example, a state in which 10 or more cells adhere to each other). It is a concept opposite to detached cells and deemed isolated cells.
  • An isolated cell refers to one cell that is in an independent state without adhering to other cells.
  • a deemed isolated cell refers to a group of several cells that are in contact with one or two other cells, or are gathered to such an extent that they are weakly attached and easily separated.
  • the cell mass refers to one mass obtained by adhering a plurality of (for example, 10 or more) cells (endoderm cells) obtained in the step (3) to each other.
  • a state where Cell aggregates, isolated cells, and deemed isolated cells may be distinguished by the number of cells that have aggregated (for example, if 3 or more cells are aggregated, the cell mass).
  • the images may be distinguished by the area of a single area as a cell.
  • the cross-sectional area is about 300 ⁇ m 2 . Therefore, for example, if the area of a single region as a cell is less than 300 ⁇ m 2, it may be an isolated cell and a deemed isolated cell, and if it is larger than 900 ⁇ m 2 (ie, 3 cells), it may be a cell mass. If 10 or more cells form a cell mass adhered to each other, the cross-sectional area is assumed to be 3000 ⁇ m 2 or more.
  • culturing in a suspended state means culturing in a medium under non-adhesive conditions.
  • Cultivation under non-adhesive conditions refers to culturing in a state where it is not adhered to an incubator or a substrate (for example, using a non-adhesive multi-well plate).
  • Cultivation under non-adhesive conditions can be performed by a method known per se. As such a method, for example, the surface of an incubator is coated with a hydrophilic substance proteoglycan such as polyhydroxyethyl methacrylate to inhibit the adhesion of cells to a substrate (Cell Struct Funct, 13, 179).
  • protein digestive enzymes are added to the cells (endoderm cells) obtained in the step (3) to separate the cells so that they are in a single cell state.
  • protein digestive enzymes include, but are not limited to, trypsin, collagenase, papain, dispase, and inactogen (trade name). These protein digestive enzymes are typically trypsin-EDTA solutions (eg, 0.25% trypsin-1 mM) with the addition of EDTA to chelate Ca 2+ and Mg 2+ , which are inhibitors of digestive enzyme action. EDTA).
  • the cell mass can be prepared from the cells obtained in the step (3) as follows, for example. That is, the cells obtained in the step (3) are suspended and cultured in an appropriate medium on an incubator. For example, when a 96-well round bottom dish is used as a low adhesion incubator, 20 to 400,000 cells are seeded per well and the formed aggregate is transferred to a low adhesion 6 cm dish or the like. However, the cells are cultured in an appropriate medium at 37 ° C. for about 6 hours to about 10 days, preferably about 6 hours to about 2 days, more preferably 1 day. Examples of the low-adhesive incubator include those obtained by treating the incubator used in this technical field so as to be low-adhesive.
  • the incubator examples include a culture dish, a culture flask, a rotary culture instrument (such as a spinner flask), and specifically a spheroid plate.
  • a treatment for achieving low adhesion a treatment for suppressing the adhesion of proteins and cells by covalently bonding hydrogel (coating) is used.
  • the cells obtained in the above step (3) are seeded at a density of 2 ⁇ 10 4 per well in a 96-well spheroid plate, for example, at 37 ° C., 5% CO 2. Under the conditions described above, the cells are cultured in Improved MEM Zinc Option medium supplemented with 1% B27 supplement for 1 day.
  • the cell mass obtained as described above is cultured in a suspended state in a medium.
  • the culture in a floating state refers to culture under non-adhesive conditions, that is, culture in a state where it is not adhered to a culture vessel or a substrate (eg, non-adhesive multiwell plate).
  • the medium used in this step include the basal medium exemplified in the step (2).
  • the medium used in this step may be prepared using the same type of basal medium as in the above steps (2) to (3), or may be prepared using a different type of basal medium.
  • Improved MEM Zinc Option medium (Invitrogen) is preferably used as the basal medium in this step in that differentiation induction into pancreatic hormone-producing cells can be performed more efficiently.
  • the medium can also be prepared according to known literature (Richter A. et al., National Cancer (1972) 49, 1705).
  • the medium used in this step may contain a serum substitute.
  • Serum substitutes include albumin, transferrin, fatty acid, collagen precursor, trace elements (eg, zinc, selenium), B27 supplement, N2 supplement, knockout serum replacement, 2-mercaptoethanol, 3'thiol glycerol, and equivalents thereof Such as things.
  • B27 supplement is preferable as a serum substitute used in this step.
  • Improved MEM Zinc Option medium (Invitrogen) supplemented with B27 supplement is particularly preferably used.
  • concentration of B27 supplement in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight.
  • substantially no feeder cells and / or feeder cell extracts are used, more preferably no feeder cells and / or feeder cell extracts are used.
  • a causative substance eg, animal-derived cells
  • a medium used in this step a medium substantially free of serum and / or serum extract is preferable, and a serum-free medium is more preferable.
  • the culture temperature is not particularly limited as long as it is a culture temperature suitable for culturing cells to be used.
  • the culture time is 6 to 360 hours, preferably about 1 day to about 12 days, and more preferably about 8 days.
  • the cell mass formed in a floating state after the cell mass is formed, the cell mass can be subjected to the step (5) without being subjected to suspension culture.
  • the cell mass formed before moving to the step (5) may be cultured in a floating state for 0 to 48 hours, preferably 0 to 24 hours.
  • the culture in this step is usually performed in an incubator in which about 1 to 10%, preferably 5% CO 2 is aerated.
  • the cells obtained in the above step (3) are seeded at a density of 2 ⁇ 10 4 per well in a 96-well spheroid plate, for example, at 37 ° C., 5% CO 2. Under the conditions described above, the cells are cultured in Improved MEM Zinc Option medium supplemented with 1% B27 supplement for 1 day.
  • pancreatic hormone-producing cells can be produced using endoderm cells other than the endoderm cells obtained through steps (1) to (3) as starting materials. Therefore, the present invention provides a method for producing pancreatic hormone-producing cells using endoderm cells as a starting material by this step (4), that is, a cell mass is formed from endoderm cells, and the cell mass is suspended in a medium.
  • the method for producing pancreatic hormone-producing cells (which may be abbreviated as production method 2 of the present invention in the present specification) is also provided.
  • the method for producing pancreatic hormone-producing cells using the endoderm cells other than the endoderm cells obtained through the above steps (1) to (3) as a starting material also includes the cells obtained in the above step (3).
  • step (4) This can be carried out in the same manner as in step (4) in the method for producing pancreatic hormone-producing cells as a starting material.
  • the production method 2 of the present invention is characterized in that the endoderm cells are subjected to the following steps (4 ′) and (5 ′).
  • Step 4 ′) A step of forming a cell mass from endoderm cells and culturing the cell mass in a suspended state in a medium (5 ′)
  • the cell obtained in (4 ′) above is treated with a retinoic acid receptor agonist, AMP Culturing in a medium containing an activated protein kinase and / or an inhibitor of activin receptor-like kinase-2,3,6, an inhibitor of activin receptor-like kinase-4,5,7, and a cell growth factor
  • Step 4 ′) can be performed in the same manner as step (4) and step (5 ′) in the same manner as step (5).
  • a pancreatic hormone-producing cell that more closely mimics the development of pancreas can be produced. Since the cells have a three-dimensional cell cluster structure, they are considered to be more functional and closer to the state in vivo than monolayer cultured cells. Furthermore, the cells are considered to be more suitable for application to cell medicine than the pancreatic hormone-producing cells obtained by the conventional production method due to the three-dimensional structure.
  • Step (5) The cells obtained in the above step (4) are treated with retinoic acid receptor agonist, AMP-activated protein kinase and / or activin receptor-like kinase-2,3,6 inhibitor, activin receptor-like Step of culturing in a medium containing an inhibitor of kinase-4, 5, 7 and cell growth factor
  • This step is performed from cells obtained through the above step (4), that is, from endoderm cells cultured in a floating state. This corresponds to the step of inducing differentiation into pancreatic hormone-producing progenitor cells.
  • the retinoic acid receptor (RAR) agonist used in this step may be a natural retinoid, a synthetic retinoid, a retinoic acid receptor agonist having no retinoid skeleton, or a natural product having equivalent activity.
  • RAR retinoic acid receptor
  • An example of a natural retinoid is retinoic acid (the stereoisomers all-trans-retinoic acid (all-trans RA) and 9-cis-retinoic acid (9-cis RA) are known).
  • Synthetic retinoids are known in the art (US Pat. No. 5,234,926, US Pat. No. 4,326,055, etc.).
  • retinoic acid receptor agonists examples include Am80, TTNPB, AC-55649, and the like.
  • natural products examples include honokiol, magnolol, etc. (Research Bulletin of Laboratory for Biofunction Development 9: 55-61, 2009).
  • the RAR agonist used in this step is preferably retinoic acid.
  • the concentration of the RAR agonist in the medium is appropriately set depending on the type of agonist used. In the case of retinoic acid, it is usually 0.1 to 100 ⁇ M, preferably 0.5 to 10 ⁇ M.
  • the inhibitor of AMP-activated protein kinase and / or activin receptor-like kinase-2, 3, 6 used in this step is a compound having AMP-activated protein kinase (AMPK) inhibitory activity, activin receptor-like kinase (ALK). It is selected from the group consisting of compounds having -2, 3, 6 inhibitory activity, and compounds having both AMP-activated protein kinase inhibitory activity and activin receptor-like kinase-2, 3, 6 inhibitory activity.
  • AMPK AMP-activated protein kinase
  • ALK activin receptor-like kinase
  • Compounds having AMPK inhibitory activity include dosomorphin (Dorsomorphin: 6- [4- (2-piperidin-1-ylethoxy) phenyl] -3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine), araA (Adenine-9- ⁇ -d-arabinofuranoside), C75 and the like.
  • Activin receptor-like kinase (ALK) is classified into several types, ALK-2, 3, and 6 are BMP type I receptor type kinases, and ALK-4, 5, and 7 described later are TGF- ⁇ superfamily type I. Are known as receptor-type kinases.
  • Compounds having ALK-2,3,6 inhibitory activity include dosomorphin, LDN-193189 (6- (4-piperazinophenyl) -3- (quinolin-4-yl) pyrazolo [1,5-a ] Pyrimidine) and the like.
  • Dosomorphin has both AMPK inhibitory activity and ALK-2,3,6 inhibitory activity.
  • These compounds can be purchased from SIGMA, Tocris bioscience, Stemgent, Merck Bioscience, and the like.
  • antisense oligonucleotides and siRNA for AMP-activated protein kinase and ALK-2,3,6 mRNA can also be used as inhibitors of AMP-activated protein kinase and / or ALK-2,3,6.
  • an antibody that neutralizes the activity of the differentiation factor, or BMP Noggin, Chordin, Cerberus, Gremlin, etc., which are known to bind to and inhibit its action can also be used as inhibitors of AMP-activated protein kinase and / or ALK-2,3,6.
  • activin when the increase or secretion of activin exemplified as the activator of activin receptor-like kinase-4, 7 in the step (3) is confirmed from the cells in culture in the medium in this step, activin is confirmed.
  • An antibody that neutralizes the activity of phosphatase or follistatin that is known to bind to activin and inhibit its action may be used as an inhibitor of AMP-activated protein kinase and / or ALK-2, 3, 6 it can.
  • the concentration of the inhibitor of AMP-activated protein kinase and / or activin receptor-like kinase-2, 3, 6 in the medium is appropriately set depending on the type of the inhibitor to be used. 1 to 20 ⁇ M, preferably 0.2 to 5 ⁇ M.
  • inhibitors of activin receptor-like kinase (ALK) -4,5,7 used in this step include SB-431542 and SB-505124 (2- (5-benzo [1,3] dioxol-5-yl-2). -Tert-butyl-3H-imidazol-4-yl) -6-methylpyridine hydrochloride), SB-525334 (6- [2-tert-butyl-5- (6-methyl-pyridin-2-yl) -1H -Imidazol-4-yl] -quinoxaline), A-83-01 (3- (6-methyl-2-pyridyl) -N-phenyl-4- (4-quinolinyl) -1H-pyrazole-1-thiocarboxamide) , GW6604, LY-580276 (2- (6-methyl-2-pyridinyl) -3- (4-fluorophenyl) -5,6-dihydro-4H-pyrrolo [1,2 b] pyrazole) and
  • antisense oligonucleotides, siRNA and the like against ALK-4,5,7 mRNA can also be used as inhibitors of ALK-4,5,7.
  • SB-431542 As an inhibitor of ALK-4,5,7 used in this step, SB-431542 (4- [4- (1,3-benzodioxol-5-yl) -5- (2-pyridinyl) -1H -Imidazol-2-yl] -benzamide or hydrates thereof.
  • concentration of the activin receptor-like kinase-4,5,7 inhibitor in the medium is appropriately set depending on the type of the inhibitor used. In the case of SB-431542, it is usually 0.1-50 ⁇ M, preferably 1 ⁇ 20 ⁇ M.
  • vascular endothelial growth factor VEGF
  • HGF hepatocyte growth factor
  • SCF stem cell growth factor
  • EGF epidermal growth factor
  • a / BFGF various fibroblast growth factors
  • the concentration of the cell growth factor in the medium is appropriately set depending on the type of factor used. In the case of bFGF, it is usually 1 to 200 ng / ml, preferably 20 to 100 ng / ml.
  • This step includes the above-described retinoic acid receptor agonist, AMP-activated protein kinase and / or activin receptor-like kinase-2,3,6 inhibitor, activin receptor-like kinase-4,5,7 inhibitor, And in a medium containing all four components of cell growth factor.
  • a retinoic acid receptor agonist an AMP-activated protein kinase and / or an activin receptor-like kinase-2,3,6 inhibitor, an activin receptor-like kinase-4,5,7 inhibitor, and a cell
  • the growth factor may be added simultaneously to the medium, or may be added to the medium separately with a time difference as long as differentiation into pancreatic hormone-producing progenitor cells can be induced.
  • a retinoic acid receptor agonist, an AMP-activated protein kinase and / or an inhibitor of activin receptor-like kinase-2,3,6, an inhibitor of activin receptor-like kinase-4,5,7, and a cell growth factor It is convenient and preferable that they are added simultaneously to the medium.
  • the medium used in this step is the same as the basal medium exemplified in the above step (2), but is an retinoic acid receptor agonist, an AMP-activated protein kinase and / or an activin receptor-like kinase-2,3,6 inhibitor, an activin receptor. It is produced by adding an inhibitor of body-like kinase-4, 5, 7 and a cell growth factor.
  • the medium used in this step may be prepared using the same type of basal medium as in step (4), or may be prepared using a different type of basal medium.
  • Improved MEM Zinc Option medium (Invitrogen) is suitably used as the basal medium in this step in that differentiation induction into pancreatic hormone-producing progenitor cells can be performed more efficiently.
  • substantially no feeder cells and / or feeder cell extracts are used, more preferably no feeder cells and / or feeder cell extracts are used.
  • a causative substance eg, animal-derived cells
  • a medium used in this step a medium substantially free of serum and / or serum extract is preferable, and a serum-free medium is more preferable.
  • the medium used in this step may contain a serum substitute.
  • Serum substitutes include albumin, transferrin, fatty acids, collagen precursors, trace elements (eg zinc, selenium), B27 supplements, N2 supplements, knockout serum replacement, 2-mercaptoethanol, 3'thiolglycerol and their equivalents Etc.
  • B27 supplement is desirable.
  • the concentration of the serum substitute in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight in the case of B27 supplement.
  • This step is performed at a culture temperature suitable for culturing endoderm cells to be used, usually 30 to 40 ° C., preferably about 37 ° C. for 72 to 288 hours, preferably 120 to 216 hours, 1 to 10%, preferably 5%. This is carried out by culturing in a CO 2 incubator aerated with carbon dioxide.
  • pancreatic hormone-producing progenitor cell markers proteins and genes that are specifically expressed in pancreatic hormone-producing progenitor cells.
  • Protein expression can be evaluated by a method using an antigen-antibody reaction, and gene expression can be evaluated by a method using RT-PCR.
  • the marker include NGN3, HNF6 (hepatocyte nuclear factor 6, alias: one cut homebox 1), PDX1 (pancreatic and dual homebox 1), and the like.
  • Step (6) A step of culturing the cells obtained in the step (5) in a medium. This step corresponds to a step of inducing differentiation from pancreatic hormone-producing precursor cells into pancreatic hormone-producing cells.
  • the basal medium used in this step includes the basal medium exemplified in the above step (2).
  • the medium used in this step may be prepared using the same type of basal medium as in step (5) or may be prepared using a different type of basal medium.
  • Improved MEM Zinc Option medium (Invitrogen) is preferably used as a basal medium in this step in that differentiation into pancreatic hormone-producing cells can be more efficiently induced.
  • substantially no feeder cells and / or feeder cell extracts are used, more preferably no feeder cells and / or feeder cell extracts are used.
  • a causative substance eg, animal-derived cells
  • a medium used in this step a medium substantially free of serum and / or serum extract is preferable, and a serum-free medium is more preferable.
  • the medium used in this step may contain a serum substitute.
  • Serum replacements include albumin (eg, lipid-rich albumin), transferrin, fatty acid, collagen precursor, trace elements (eg, zinc, selenium), B27 supplement, N2 supplement, knockout sealum replacement, 2-mercaptoethanol or 3 ′ Examples thereof include thiol glycerol and equivalents thereof.
  • B27 supplement is preferable.
  • Improved MEM Zinc Option medium (Invitrogen) supplemented with B27 supplement is preferably used.
  • the concentration of B27 supplement in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight.
  • examples of such additives include serum substitutes such as knockout sealant replacement and N2 supplement.
  • the concentration of the additive in the medium is 0.01 to 10% by weight, preferably 0.1 to 2% by weight.
  • the medium used in this step comprises a retinoic acid receptor agonist, an AMP-activated protein kinase and / or an inhibitor of activin receptor-like kinase-2,3,6, activin receptor-like kinase-4,5, 7 inhibitors and cell growth factors are not included. Therefore, it is preferable to exchange the medium between step (5) and step (6).
  • This step is performed at a culture temperature suitable for culturing pancreatic hormone-producing precursor cells to be used, usually 30 to 40 ° C., preferably about 37 ° C. for 24 to 240 hours, preferably 72 to 192 hours, 1 to 10%, preferably It is carried out by culturing in a CO 2 incubator aerated with 5% carbon dioxide. Confirmation that pancreatic hormone-producing progenitor cells were induced to differentiate into pancreatic hormone-producing cells in this step is the expression of proteins and genes (pancreatic hormone-producing cell markers) that are specifically expressed in pancreatic hormone-producing cells, or secretion into the medium. This can be done by measuring the amount of pancreatic hormones produced.
  • markers of the marker include insulin, glucagon, pancreatin polypeptide, somatostatin, ghrelin, PCSK1 (protein convertase subtilisin / kexin type 1), SUR1 (sulfonylurea receptor 1, alias: ATP-bindte MRP), member 8), NKX6.1 (NK6 homebox 1), PAX6 (paired box 6), NEUROD (neurogenous differential 1), ARX (aristrals related home) and the like.
  • the present invention provides a method for producing pancreatic hormone-producing cells from stem cells.
  • stem cells are produced by the same method, that is, a method for inducing differentiation of cells in a more undifferentiated state into a more differentiated state.
  • differentiation can be induced into cells in various differentiated states (endoderm cells, pancreatic duct cells, pancreatic endocrine cells, exocrine pancreatic cells, progenitor cells common to them, and the like).
  • the degree of differentiation induced can be determined by confirming the presence or absence of expression of a protein or gene specifically expressed in each cell.
  • pancreatic hormone-producing cells having high pancreatic hormone-secreting ability can be supplied by efficiently inducing differentiation of stem cells into pancreatic hormone-producing cells.
  • This pancreatic hormone-producing cell can be used as a drug (particularly a drug for cell therapy) or a tool for developing a therapeutic drug for diabetes.
  • the present invention provides a medicament comprising pancreatic hormone-producing cells (cells of the present invention) produced by the production method of the present invention or the production method 2 of the present invention described above. Furthermore, the present invention relates to a medicament comprising pancreatic hormone-producing progenitor cells produced by the production method of the present invention described above (preferably steps (1) to (5)) or the production method 2 of the present invention (in the present specification, (It may be abbreviated as the pharmaceutical of the present invention).
  • pancreatic hormone-producing cells or pancreatic hormone-producing precursor cells are used as they are or as a cell mass such as a pellet concentrated by filter filtration or the like. Furthermore, the medicament can be stored frozen by adding a protective agent such as DMSO (dimethyl sulfoxide).
  • DMSO dimethyl sulfoxide
  • the protein of the pathogen is denatured while leaving the function as a pancreatic hormone-producing cell or the function as a pancreatic hormone-producing progenitor cell, such as heat treatment and radiation treatment. You may attach to the process on conditions.
  • pancreatic hormone-producing cells or pancreatic hormone-producing progenitor cells in order to prevent pancreatic hormone-producing cells or pancreatic hormone-producing progenitor cells from growing beyond the necessary amount, in combination with the above treatment, suppression of proliferation by mitomycin C pretreatment, etc., and mammals naturally have. Introduce a gene for a non-metabolizing enzyme into the cell, then administer an inactive drug as needed, and the drug only in the cell into which the gene for the metabolic enzyme that the mammal does not naturally have is introduced. It may be subjected to a treatment such as a method of killing cells by changing to a poison (suicide gene therapy).
  • the medicament of the present invention is safe and has low toxicity, and can be administered (transplanted) to a mammal (for example, human, mouse, rat, guinea pig, pig, monkey, etc., preferably human).
  • the dose (transplant amount) of the medicament of the present invention is, for example, 1 ⁇ 10 5 to 1 ⁇ 10 10 cells / individual, preferably 5 ⁇ 10 7 to 1 ⁇ 10 10 cells / individual, more preferably 1 ⁇ 10 9 to 1 ⁇ 10 10 cells / individual.
  • pancreatic hormone-producing cells prepared using the patient's own cells or histocompatibility-acceptable donor cells, but sufficient cells are obtained for reasons such as age and constitution.
  • the medicament of the present invention can be implanted in a state where a rejection reaction is avoided by embedding it in a capsule such as polyethylene glycol or silicone, or in a porous container. In such a case, implantation into the abdominal cavity or subcutaneous is also possible. Further, the dose (transplant amount) of the medicament of the present invention can be appropriately changed depending on the age, weight, symptoms, etc. of the patient to be administered.
  • the medicament containing pancreatic hormone-producing cells can be produced (secreted) in the body of the patient by administration (transplantation) itself, and the production (secretion) of pancreatic hormone can be reduced.
  • a medicament comprising insulin-producing cells is useful for the treatment of diabetes (types I and II, preferably type I or type II with reduced insulin production, particularly preferably type I).
  • a drug containing pancreatic hormone-producing progenitor cells is administered (transplanted) to a patient and then induced to differentiate into pancreatic hormone-producing cells under appropriate conditions, thereby producing pancreatic hormones. (Secreted).
  • Screening method uses a cell obtained by any one or more steps selected from the group consisting of the following steps (1) to (6), preferably a medicament (preferably a therapeutic agent for diabetes) ) Screening method (which may be abbreviated as the screening method of the present invention in the present specification).
  • Step of culturing stem cells in medium containing Rho kinase inhibitor (2) Step of culturing cells obtained in (1) above in medium containing GSK3 inhibitor (3) Obtained in (2) above A step of culturing the obtained cells in a medium containing a GSK3 inhibitor and an activin receptor-like kinase-4,7 activator (4) After forming a cell mass from the cells obtained in (3) above Culturing the cell mass in a suspended state in a medium (5)
  • the cells obtained in the above (4) are retinoic acid receptor agonist, AMP-activated protein kinase and / or activin receptor-like kinase-2, A step of culturing in a medium comprising an inhibitor of 3, 6; an inhibitor of activin receptor-like kinase-4, 5, 7; and a cell growth factor (6) culturing the cell obtained in (5) above in the medium Process
  • the steps (1) to (6) can be carried out in the same manner as the steps (1) to (6) in the above-described method for producing pancreatic hormone-producing cells of the present invention.
  • the cells used in this screening include pancreatic hormone-producing cells obtained through the above steps (1) to (6); pancreatic hormone-producing progenitor cells obtained through the above steps (1) to (5); ) To (4) or the endoderm cells obtained through the above steps (1) to (3); the cells obtained through the above steps (1) to (2); and the cells obtained through the above step (1). It is done.
  • the screening method of the present invention is carried out as follows (Aspect 1). (A) when pancreatic hormone-producing cells are cultured in the presence of a test compound, and (b) when pancreatic hormone-producing cells are cultured in the absence of a test compound, A method of measuring and comparing pancreatic hormone secretion amounts of each of them.
  • the expression level of pancreatic hormone include the expression level of pancreatic hormone protein, the expression level of a polynucleotide (eg, mRNA) encoding pancreatic hormone protein, and the like.
  • Measurement of pancreatic hormone expression level and secretion level is a known method, for example, using an antibody recognizing pancreatic hormone, the pancreatic hormone present in a cell extract or medium, Western blotting analysis, ELISA method It is performed according to the method or the method according to it.
  • the amount of mRNA is measured according to a known method, for example, Northern hybridization, S1 mapping method, PCR method, DNA chip or array method, or a similar method.
  • Culture of pancreatic hormone-producing cells is not particularly limited as long as pancreatic hormone is expressed and / or secreted, and may be performed according to a known method.
  • Examples of the medium include MEM medium containing about 1 to 20% fetal bovine serum [Science, 122, 501 (1952), etc.], DMEM medium [Virology, 8, 396 (1959)], RPMI 1640 medium [ The Journal of the American Medical Association 199, 519 (1967)], 199 medium [Proceeding of the Society for the Biological Medicine, 73, 1 (1950)] is used.
  • the pH of the medium is preferably about 6-8.
  • the culture is usually performed at about 30 ° C. to 40 ° C. for about 15 hours to 5 days, and aeration and agitation are added as necessary.
  • test compound examples include peptides, proteins, antibodies, non-peptide compounds, synthetic compounds, fermentation products, cell extracts, plant extracts, animal tissue extracts, and plasma.
  • the test compound may form a salt.
  • the salt include salts with physiologically acceptable acids and bases (eg, alkali metal salts, alkaline earth metal salts, aluminum salts).
  • salts include inorganic acids ( For example, salts with hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), organic acids (eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid) Acid, methanesulfonic acid, benzenesulfonic acid), sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, and aluminum salts.
  • inorganic acids For example, salts with hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid
  • organic acids eg acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, succinic acid, benzoic acid
  • Acid methanesulfonic acid, benzenesulfonic acid
  • sodium salts potassium salts
  • the expression level or secretion level of pancreatic hormone in the case (a) is suppressed by about 20% or more, preferably about 30% or more, more preferably about 50% or more compared to the case (b)
  • the test compound to be inhibited can be selected as a compound that suppresses (inhibits) pancreatic hormone expression in pancreatic hormone-producing cells.
  • Test compound that promotes the expression level or secretion level of pancreatic hormone in the case of (a) above about 20% or more, preferably about 30% or more, more preferably about 50% or more, compared to the case of (b) above Can be selected as a compound that promotes pancreatic hormone expression or secretion in pancreatic hormone-producing cells.
  • pancreatic hormone-producing cell When the pancreatic hormone-producing cell is an insulin-producing cell, a compound that promotes insulin expression is useful as a therapeutic agent for diabetes.
  • the pancreatic hormone-producing cell is a glucagon-producing cell, a compound that suppresses (inhibits) glucagon expression is useful as a therapeutic agent for diabetes.
  • a method for measuring the number of cells, 3 H, 5-bromo-2′-deoxy-uridine (BrdU) And the like, the amount of ATP, and the amount of conversion from a tetrazolium compound to a formazan product can be evaluated.
  • the pancreatic hormone-producing cell is an insulin-producing cell
  • a compound that significantly promotes the growth of the insulin-producing cell is useful as a therapeutic agent for diabetes.
  • the pancreatic hormone-producing cell is a glucagon-producing cell
  • a compound that significantly suppresses (inhibits) the growth of the glucagon-producing cell is useful as a therapeutic agent for diabetes.
  • Another embodiment of the screening method of the present invention is as follows: (a) when pancreatic hormone-producing progenitor cells are cultured in the presence of the test compound; and (b) when pancreatic hormone-producing progenitor cells are cultured in the absence of the test compound. And a method of examining and comparing the degree of differentiation of the cells, respectively (Aspect 3).
  • the test compound to be used include the same compounds as those used in the first aspect.
  • the cell culture in this embodiment can be performed in the same manner as in Embodiment 1 above.
  • pancreatic hormone-producing progenitor cells The degree of differentiation of pancreatic hormone-producing progenitor cells is examined by, for example, the presence or absence of expression of a specific marker of pancreatic hormone-producing progenitor cells and / or pancreatic hormone-producing cells.
  • Specific markers for pancreatic hormone-producing progenitor cells are NGN3 (neurogenin 3) and PAX4 (paired box 4), and specific markers for pancreatic hormone-producing cells are insulin, glucagon, pancreatic polypeptide, somatostatin, ghrelin, PCSK1 ( proprotein convertase subtilisin / kexin type 1), SUR1 (sulfonylurea receptor 1, also known as ATP-binding cassette, sub-family C (CFTR / MRP), member 8), glucokinase, MAFA (v-maf musculoaponeurotic fibrosarcoma oncogene homolog A), IAPP (islet am yloid polypeptide
  • pancreatic hormone-producing progenitor cell when the pancreatic hormone-producing progenitor cell is an insulin-producing progenitor cell, a compound that significantly promotes differentiation of the insulin-producing progenitor cell is useful as a therapeutic agent for diabetes.
  • the pancreatic hormone-producing progenitor cell is a glucagon-producing progenitor cell, a compound that significantly suppresses (inhibits) the differentiation of the glucagon-producing progenitor cell is useful as a therapeutic agent for diabetes.
  • Another embodiment of the screening method of the present invention is such that (a) when endoderm cells are cultured in the presence of the test compound, and (b) when endoderm cells are cultured in the absence of the test compound.
  • a method of measuring and comparing proliferation or differentiation ability is mentioned (Aspect 4). Examples of the test compound to be used include the same compounds as those used in the first aspect.
  • the cell culture in this embodiment can be performed in the same manner as in Embodiment 1 above.
  • a method for measuring the proliferation ability of a cell a method practiced in the art is usually used.
  • a method for measuring the number of cells, 3 H, 5-bromo-2′-deoxy-uridine (BrdU) And the like, the amount of ATP, and the amount of conversion from a tetrazolium compound to a formazan product can be evaluated.
  • the differentiation ability of endoderm cells is examined by, for example, the presence or absence of expression of a specific marker of endoderm cells.
  • Specific markers for endoderm cells include alpha fetoprotein, albumin, pepsin, pulmonary surfactant protein, and the like.
  • differentiation induction and culture of endoderm cells are technically difficult compared to mesoderm or ectoderm cells, and the cells themselves and / or endoderm prepared using the compounds obtained by the screening system
  • the differentiation induction system can be used for a new drug screening system.
  • a medicine or the like that protects (maintains) the function of pancreatic hormone-producing cells can also be obtained by a method according to the screening method of the present invention.
  • the screening method of the present invention (a) when pancreatic hormone-producing cells are cultured in the presence of a test compound, and (b) when pancreatic hormone-producing cells are cultured in the absence of the test compound, A method of measuring and comparing the number of viable cells or their functions, respectively (Aspect 5).
  • the test compound to be used include the same compounds as those used in the first aspect.
  • the cell culture in this embodiment can be performed in the same manner as in Embodiment 1 above.
  • a method for measuring the number of viable cells a method practiced in the art is usually used.
  • a method for measuring the number of cells or 3 H 5-bromo-2′-deoxy-uridine (BrdU) And the like, the amount of ATP, and the amount of conversion from a tetrazolium compound to a formazan product.
  • the number of cells in which apoptosis is induced is determined by the number of cells exhibiting morphological characteristics (condensation of chromatin, nuclear fragmentation, cell contraction, etc.), as well as TUNNEL (TdT-mediated dUTP nick labeling).
  • Examples of the method for measuring cell function include a method for measuring changes in insulin or C peptide secretion and cell membrane potential according to the glucose concentration.
  • factors known to damage pancreatic hormone-producing cells for example, inflammatory cytokines, active oxygen and their production inducers, high concentrations of fatty acids and glucose are added during cell culture.
  • pancreatic hormone-producing cell is an insulin-producing cell
  • the compound that significantly promotes the survival or maintenance of the function of the insulin-producing cell against a factor known to damage the pancreatic hormone-producing cell is a therapeutic agent for diabetes As useful.
  • the medicament obtained using the screening method of the present invention is formulated using physiologically acceptable additives (eg, carrier, flavoring agent, excipient, preservative, stabilizer, binder) according to conventional means.
  • physiologically acceptable additives eg, carrier, flavoring agent, excipient, preservative, stabilizer, binder
  • Can be Examples of the dosage form of the preparation thus obtained include oral preparations such as tablets, capsules, elixirs and microcapsules with sugar coating as required; and parenteral preparations such as injections.
  • the content of the active ingredient in these preparations is, for example, 0.1 to 90% by weight.
  • the additive examples include binders such as gelatin, corn starch, tragacanth and gum arabic; excipients such as crystalline cellulose; swelling agents such as corn starch, gelatin and alginic acid; lubricants such as magnesium stearate; sucrose Sweeteners such as lactose and saccharin; flavoring agents such as peppermint, red oil and cherry; oils and fats, water for injection, vegetable oils (eg sesame oil, coconut oil, soybean oil), buffers (eg phosphate buffer, acetic acid Liquid carriers such as sodium buffer; solubilizers (eg, ethanol, propylene glycol, polyethylene glycol); nonionic surfactants (eg, polysorbate 80TM, HCO-50); solubilizers (eg, benzyl benzoate) Benzyl alcohol); soothing agents (eg, benzalco chloride) Um, procaine hydrochloride); stabilizers (e.g., human serum albumin, polyethylene glycol); preserv
  • the water for injection examples include physiological saline; isotonic solutions containing glucose, D-sorbitol, D-mannitol, sodium chloride and the like.
  • the medicament preferably antidiabetic agent obtained by the screening method of the present invention is safe and low toxic, for example, mammals (eg, humans, mice, rats, rabbits, sheep, pigs, cows, horses, cats, Canine, monkey, chimpanzee) orally or parenterally.
  • the dosage of the drug is appropriately determined depending on its action, target disease, administration subject, administration route and the like.
  • Reprocell primate ES cell detachment solution
  • a 0.25% trypsin-1 mM EDTA solution (GIBCO) was added to the human iPS cells that had settled in the centrifuge tube, and dissociated until they became single cells.
  • human iPS cells dispersed in the medium were seeded at a density of 15 ⁇ 10 4 per 100 mm dish coated with BD Matrigel (Nippon Becton Decktonson), and at 37 ° C. under 5% CO 2 . Cultured for 1 day. A 100 mm dish was prepared by diluting BD Matrigel 40 times using serum-free DMEM / F12 medium (Invitrogen) and coating at room temperature for 3 hours or more.
  • a medium for primate ES cells (Reprocell) supplemented with 10 ⁇ M Y-27632 (Wako Pure Chemical Industries) was used.
  • the medium was changed to a DMEM / F12 medium containing CHIR99021 (3 ⁇ M), a GSK3 ⁇ inhibitor and 2% B27 supplement (GIBCO), and cultured for 2 days.
  • the medium was changed to a DMEM / F12 medium containing activin A (50 ng / ml) and CHIR99021 (1 ⁇ M), and cultured for 2 days.
  • the differentiation-induced cells were collected over time, and the total RNA fraction was purified using RNeasy 96 (Qiagen). After synthesizing cDNA using PrimeScript RT reagent kit (Takara Bio Inc.), quantitative RT-PCR was performed to measure the gene expression level of Brachyury as a primitive streak marker and SOX17 as an endoderm marker. The results of expression analysis are shown in FIG. The expression level of Brachyury was transiently increased on the third day of culture, and the expression level of SOX17 was significantly increased on the fourth day. This revealed that the endoderm marker was efficiently induced to induce expression through primitive streak.
  • the cells were cultured for 2 days in DMEM / F12 medium supplemented with CHIR99021 and B27 supplements, and further in DMEM / F12 medium supplemented with activin A and CHIR99021 for 2 days without using feeder cells or serum. It was revealed that differentiation into endoderm cells can be induced.
  • Example 1 (2) The endoderm induction by GSK3 inhibitors (BIO) other than CHIR99021 in the step (2) was examined.
  • BIO GSK3 inhibitors
  • the medium was replaced with DMEM / F12 medium containing CHIR99021 (3 ⁇ M) and 2% B27 supplement (GIBCO) and cultured for 2 days.
  • DMEM / F12 medium containing CHIR99021 (3 ⁇ M)
  • activin A 100 ng / ml
  • 2% B27 supplement 2% B27 supplement
  • Example 2 Differentiation induction from endoderm cells to pancreatic hormone-producing cells [steps (4) to (6)] A cell mass was formed from the cells differentiated into endoderm cells, and then further differentiated into pancreatic hormone-producing progenitor cells, and subsequently into pancreatic hormone-producing cells. Endoderm cells induced to differentiate using a 100 mm dish were dissociated using Invitrogen (trade name) until they became single cells. Subsequently, the endoderm cells dispersed in the medium were seeded in a 96-well spheroid plate (Sumitomo Bakelite) at a density of 2 ⁇ 10 4 per well and cultured at 37 ° C. under 5% CO 2 for 1 day. A lump was formed.
  • a 96-well spheroid plate Suditomo Bakelite
  • Improved MEM Zinc Option medium containing 1% B27 supplement was used as a medium for dispersion and seeding.
  • Implanted MEM Zinc Option medium containing 1% B27 supplement containing dosomorphin (1 ⁇ M), retinoic acid (2 ⁇ M), SB431542 (10 ⁇ M) and bFGF (50 ng / ml) was added.
  • the medium was changed, and the cells were cultured under the conditions of 37 ° C. and 5% CO 2 for 8 days.
  • the medium was changed once at the fourth day.
  • the medium was changed to Improved MEM Zinc Option medium containing 1% B27 supplement, and further culturing was performed.
  • the antibody is reacted with an anti-insulin antibody (A0564, DAKO) or an anti-glucagon antibody (G2654, SIGMA) as the primary antibody, and as the secondary antibody, Alexa568-labeled secondary antibody (Invitrogen) or Alexa488-labeled secondary antibody (Then, cell nuclei were stained with Hoechst 33342 and observed with a fluorescence microscope. The result of immunofluorescence staining is shown in FIG. Many cells expressing insulin were observed inside the sphere, and some cells expressing glucagon were also observed.
  • Example 3 Induction of differentiation from human iPS cells to pancreatic hormone-producing cells [Steps (1) to (6); Induction of differentiation into endoderm cells using BD Matrigel or fibronectin and induction of differentiation into pancreatic hormone-producing cells ]
  • Differentiation induction from human iPS cells to endoderm cells was performed by the following method. First, human iPS cells maintained in a cell mass state were dissociated by the same method as in Example 1 until they became single cells. Next, on the other hand, human iPS cells dispersed in a medium were seeded at a density of 60 ⁇ 10 4 per 100 mm dish coated with fibronectin (Invitrogen), and 1 at 37 ° C. under 5% CO 2. Cultured for days.
  • a 100 mm dish was prepared by diluting fibronectin 40 times with serum-free DMEM / F12 medium (Invitrogen) and coating at room temperature for 3 hours or more.
  • the other is seeded with human iPS cells dispersed in a medium at a density of 15 ⁇ 10 4 per 100 mm dish coated with BD Matrigel (Nippon Becton Decktonson), at 37 ° C. under 5% CO 2.
  • BD Matrigel Nippon Becton Decktonson
  • a medium for primate ES cells (Reprocell) supplemented with 10 ⁇ M Y-27632 (Wako Pure Chemical Industries) was used.
  • the medium was changed to a DMEM / F12 medium containing CHIR99021 (3 ⁇ M), a GSK3 ⁇ inhibitor and 2% B27 supplement (GIBCO), and cultured for 2 days.
  • the medium was changed to a DMEM / F12 medium containing activin A (50 ng / ml) and CHIR99021 (1 ⁇ M), and cultured for 2 days.
  • a cell mass was formed from the cells differentiated into endoderm cells in the same manner as in Example 2, and then further induced to differentiate into pancreatic hormone-producing progenitor cells and then pancreatic hormone-producing cells.
  • the cells were cultured with a combination of dosomorphin, retinoic acid, SB431542 and bFGF for 8 days, and then the medium was replaced with Implanted MEM Zinc Option medium containing 1% B27. Differentiation was induced and expression analysis of various differentiation markers over time was performed. The results of expression analysis are shown in FIG.
  • BD Matrigel is a basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.
  • EHS Engelbreth-Holm-Swarm
  • fibronectin used in this example was extracted from human plasma by affinity chromatography. From this, it became clear that even when fibronectin was used, differentiation into pancreatic hormone-producing cells could be induced as in the case of BD Matrigel (Examples 1 and 2).
  • pancreatic hormone-producing cells were formed using endoderm cells and cultured for 8 days in Improved MEM Zinc Option medium containing 1% B27 supplement supplemented with dosomorphin, retinoic acid, SB431542 and bFGF. Then, it was revealed that differentiation into pancreatic hormone-producing cells can be induced by using the pancreatic differentiation inducing system shown in FIG. 8 that is replaced with cultured MEM Zinc Option medium containing 1% B27 supplement and cultured. . Pancreatic hormone-producing cells produced by the method of the present invention are superior in the amount of insulin produced than pancreatic hormone-producing cells produced by other methods.
  • pancreatic hormone-producing cells that mimic pancreas development can be produced from stem cells.
  • the cells of the present invention can also be used for screening for compounds useful for the prevention and / or treatment of diseases caused by abnormal pancreatic hormone production and / or secretion such as diabetes.
  • the cells of the present invention can be used for cell therapy for treating such diseases, they maintain a three-dimensional structure, so that they are compared with pancreatic hormone-producing cells obtained by conventional production methods. Even so, it can be said that the cell is more suitable for application to cell medicine.
  • This application is based on Japanese Patent Application No. 2010-178523 for which it applied in Japan, The content is altogether included in this application.

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  • Gastroenterology & Hepatology (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Rheumatology (AREA)

Abstract

La présente invention concerne un procédé de production de cellules produisant une hormone pancréatique qui ont une forme plus semblable à celle de cellules pancréatiques qui se développent normalement à partir de cellules souches. Le procédé de production de cellules produisant une hormone pancréatique se caractérise par le fait de soumettre les cellules souches aux étapes suivantes (1)-(6) : (1) une étape de culture des cellules souches dans un milieu de culture qui contient un inhibiteur de Rho kinase ; (2) une étape de culture des cellules obtenues dans l'étape (1) dans un milieu de culture contenant un inhibiteur de GSK3 ; (3) une étape de culture des cellules obtenues dans l'étape (2) dans un milieu de culture contenant un inhibiteur de GSK3 et un agent qui active les kinases 4 et 7 de type récepteur d'activine ; (4) une étape de formation d'un agrégat cellulaire composé des cellules obtenues dans l'étape (3) et de culture dudit agrégat cellulaire dans un état flottant dans un milieu de culture ; (5) une étape de culture des cellules obtenues dans l'étape (4) dans un milieu de culture contenant un agoniste de récepteur d'acide rétinoïque, une protéine kinase activée par l'AMP et/ou un inhibiteur de kinase 2, 3 et 6 de type récepteur d'activine, un inhibiteur de kinase 4, 5 et 7 de type récepteur d'activine et un facteur de croissance cellulaire ; et (6) une étape de culture des cellules obtenues dans l'étape (5) dans un milieu de culture.
PCT/JP2011/068487 2010-08-09 2011-08-08 Procédé de production de cellules produisant une hormone pancréatique WO2012020845A1 (fr)

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US13/814,878 US9157069B2 (en) 2010-08-09 2011-08-08 Method of producing pancreatic hormone-producing cells
NZ607608A NZ607608A (en) 2010-08-09 2011-08-08 Method of producing pancreatic hormone-producing cells
SG2013007455A SG187655A1 (en) 2010-08-09 2011-08-08 Method of producing pancreatic hormone-producing cells
CA2807935A CA2807935C (fr) 2010-08-09 2011-08-08 Procede de production de cellules produisant une hormone pancreatique
CN201180048849.0A CN103154240B (zh) 2010-08-09 2011-08-08 制备胰腺激素产生细胞的方法
AU2011290123A AU2011290123B2 (en) 2010-08-09 2011-08-08 Method of producing pancreatic hormone-producing cells
KR1020137006027A KR101877077B1 (ko) 2010-08-09 2011-08-08 췌호르몬-생성 세포의 제조 방법
RU2013109949/10A RU2576000C2 (ru) 2010-08-09 2011-08-08 Способ получения клеток, продуцирующих панкреатические гормоны
JP2012528721A JP5875517B2 (ja) 2010-08-09 2011-08-08 膵ホルモン産生細胞の製造法
EP11816507.5A EP2604685B1 (fr) 2010-08-09 2011-08-08 Procédé de production de cellules produisant une hormone pancréatique
IL224451A IL224451A (en) 2010-08-09 2013-01-28 A method for producing cells that produce a pancreatic hormone

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AU (1) AU2011290123B2 (fr)
CA (1) CA2807935C (fr)
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CN103154240B (zh) 2016-09-07
CA2807935C (fr) 2018-09-18
AU2011290123A1 (en) 2013-03-07
JP5875517B2 (ja) 2016-03-02
US9157069B2 (en) 2015-10-13
RU2013109949A (ru) 2014-09-20
SG187655A1 (en) 2013-03-28
AU2011290123B2 (en) 2016-11-10
RU2576000C2 (ru) 2016-02-27
IL224451A (en) 2017-11-30
JPWO2012020845A1 (ja) 2013-10-28
EP2604685A4 (fr) 2014-03-19
KR20130099035A (ko) 2013-09-05
EP2604685B1 (fr) 2017-03-15
EP2604685A1 (fr) 2013-06-19
CA2807935A1 (fr) 2012-02-16
NZ607608A (en) 2014-03-28

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